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PathScan® Total Met Sandwich ELISA Kit #7242

Figure 1: Non-phospho and phospho Met proteins from untreated and HGF-treated A431 cells can be detected by PathScan® Total Met Sandwich ELISA kit #7242 with similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blots using Met Mouse mAb #3127 (left panel) or Met (Tyr1234/1235) Rabbit mAb #3129 (right panel), are shown in the bottom figure.

Figure 2: The relationship between protein concentration of lysates from untreated and HGF-treated A431 cells and kit assay optical density readings is shown. After starvation, A431 cells (85% confluence) were treated with HGF (40 ng/ml) for 5 min at 37°C, and then lysed.

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein is shown. Lysates were prepared from human A431 cells and incubated in wells coated with a Met Mouse mAb. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. A431 lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using a Met Rabbit antibody. A single band corresponding to the Met protein is detected in the captured material (lane 2).

Gallery: PathScan® Total Met Sandwich ELISA Kit #7242

Figure 1: Non-phospho and phospho Met proteins from untreated and HGF-treated A431 cells can be detected by PathScan® Total Met Sandwich ELISA kit #7242 with similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blots using Met Mouse mAb #3127 (left panel) or Met (Tyr1234/1235) Rabbit mAb #3129 (right panel), are shown in the bottom figure.

Figure 2: The relationship between protein concentration of lysates from untreated and HGF-treated A431 cells and kit assay optical density readings is shown. After starvation, A431 cells (85% confluence) were treated with HGF (40 ng/ml) for 5 min at 37°C, and then lysed.

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein is shown. Lysates were prepared from human A431 cells and incubated in wells coated with a Met Mouse mAb. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. A431 lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using a Met Rabbit antibody. A single band corresponding to the Met protein is detected in the captured material (lane 2).

Protocol

ELISA Colorimetric (Lyophilized)

A. Solutions and Reagents

NOTE: Prepare solutions with purified water.

Microwell strips: Bring all to room temperature before use.

Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.

HRP-Linked Antibody*: Supplied lyophilized as a red colored cake or powder. Add 1.0 ml of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.

Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

C. Test Procedure

After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4°C immediately.

Cell lysates can be undiluted or diluted with Sample Diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical kit assay results across a range of lysate concentration points.

Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.

Gently remove the tape and wash wells:

Discard plate contents into a receptacle.

Wash 4 times with 1X Wash Buffer, 200 µl each time for each well.

For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.

Clean the underside of all wells with a lint-free tissue.

Add 100 µl of reconstituted Detection Antibody (green color) to each well (refer to Section A, Step 2). Seal with tape and incubate the plate at 37°C for 1 hr.

Repeat wash procedure (Section C, Step 4).

Add 100 µl of reconstituted HRP-Linked secondary antibody (red color) to each well (refer to Section A, Step 3). Seal with tape and incubate the plate for 30 min at 37°C.

Repeat wash procedure (Section C, Step 4).

Add 100 µl of TMB Substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.

Add 100 µl of STOP Solution to each well. Shake gently for a few seconds.

NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

Product Description

CST's PathScan® Total Met Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Met protein. A Met Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Met proteins are captured by the coated antibody. Following extensive washing, Met Rabbit Antibody is added to detect both the captured phospho- and nonphospho-Met protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Met protein.

Specificity / Sensitivity

phospho- and nonphospho-Met proteins from untreated and HGF-treated A431 cell lysates are detected by this kit. In Figure 3, Western blot analysis of protein captured in the Met Mouse mAb coated microwell shows a single band corresponding to the Met protein. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Background

Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor) is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl, and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. The addition of a phosphate at cytoplasmic Tyr1003 is essential for Met protein ubiquitination and degradation (4). Phosphorylation at Tyr1234/1235 in the Met kinase domain is critical for kinase activation. Phosphorylation at Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Research studies have shown that altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon, and breast. Thus, investigators have concluded that Met is an attractive potential cancer therapeutic and diagnostic target (6,7).